The combination of Angelica sinensis polysaccharide and cisplatin promotes ferroptosis in cisplatin-resistant ovarian cancer cells by regulating GPX4, thereby reversing their resistance to cisplatin

The combination of Angelica sinensis polysaccharide and cisplatin promotes ferroptosis in cisplatin-resistant ovarian cancer cells by regulating GPX4, thereby reversing their resistance to cisplatin | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The combination of Angelica sinensis polysaccharide and cisplatin promotes ferroptosis in cisplatin-resistant ovarian cancer cells by regulating GPX4, thereby reversing their resistance to cisplatin Weikang Guo, Wanyue Wang, Fei Lei, Ruxin Zheng, Xinyao Zhao, Yuze Gu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3312243/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Angelica sinensis (Oliv.) Diels has been used for centuries in Chinese traditional medicine to treat gynecological ailments. Numerous studies indicate that Angelica sinensis polysaccharide (ASP), an extract from Angelica sinensis, can inhibit various forms of cancer. Nevertheless, the therapeutic potential of ASP for treating ovarian cancer remains largely unexplored. Methods The study investigated cell proliferation and invasion, as well as the effects of a combination of ASP and DDP after tumor formation in nude mice. Tumor size was monitored, and HE staining was conducted on tissue samples. The identification of key gene GPX4 was performed via RNA-seq and bioinformatic analysis. GPX4 was overexpressed using lentivirus transfection, and its expression was evaluated via RT-qPCR and western blot. Additionally, the ferroptosis of cells was assessed through the measurement of Fe 2+ , malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH). Results The results indicated a synergistic effect of ASP combined with DDP, leading to better inhibition of proliferation and invasion of SKOV3/DDP cells. Similarly, ASP combined with DDP demonstrated tumor proliferation inhibition in tumor-forming nude mice, while maintaining good safety. Bioinformatics analysis of 843 differentially expressed genes (DEGs) revealed that the key gene GPX4 played a significant role in the mechanism of action. Furthermore, the expression of GPX4 was inhibited by ASP combined with DDP, which resulted in SKOV3/DDP inhibition of proliferation and invasion. The study also demonstrated that ASP combined with DDP led to increased levels of Fe 2+ and MDA, while decreasing levels of GSH and SOD, suggesting the promotion ferroptosis of SKOV3/DDP cells. Conclusions The combination of ASP and DDP has the ability to inhibit the proliferation and invasion of SKOV3/DDP cells. Additionally, inhibiting GPX4 expression in SKOV3/DDP cells promotes ferroptosis. ovarian cancer traditional Chinese medicine angelica sinensis polysaccharide (ASP) GPX4 ferroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Ovarian cancer is among the top three malignancies affecting women[1]. Ovarian cancer is often diagnosed at a later stage due to the absence of typical early symptoms[2]. Cytoreductive surgery and postoperative platinum-based chemotherapy remain the standard treatments for ovarian cancer[1]. While medical technology has advanced and initial treatment outcomes for ovarian cancer have improved, many patients eventually develop chemotherapy resistance, leading to tumor recurrence and metastasis. Cisplatin (DDP) is the representative drug that patients tend to develop resistance to[3]. Hence, investigating the signaling pathways and targeted drugs tied to multi-drug resistance in ovarian cancer and, subsequently, addressing the phenomenon of multi-drug resistance, is critical for enhancing patient survival rates[4]. Angelica sinensis (Oliv.) Diels is a classic Chinese medicine prescription that has been utilized for the treatment of various gynecological diseases for thousands of years in China[5]. Angelica sinensis polysaccharide (ASP) is a major extracted component of Angelica sinensis[6]. ASP enhances the immune function of the body and acts as an immunomodulator[7], and possesses antioxidant abilities that help protect the liver[8], heart[9], and kidney[10]. Recent studies have found that ASP exhibits an antitumor effect and may aid in treating lung, liver, breast[11], and cervical cancers[12].In addition, in order to make ASP have a higher therapeutic effect on tumors, researchers analyzed ASP using 1H-NMR and FT-IR spectroscopy and created a hypoxia-responsive nano-drug delivery system relying on ASP[13, 14]. However, the effect of ASP on ovarian cancer has not been reported. In this study, we investigated the in vitro effects of ASP on SKOV3 ovarian cancer cells, as well as cisplatin-resistant SKOV3/DDP cells. Our experiments demonstrated that ASP combined with DDP can effectively reverse the resistance of SKOV3/DDP cells to DDP. We also verified the efficacy of ASP combined with DDP against tumors in nude mice, as well as evaluated the safety of ASP in vivo. Furthermore, we utilized RNA-seq data to identify differentially expressed genes (DEGs) and analyzed them to identify key genes. The effects of these key genes on cell function were subsequently verified through experiments. Materials and methods Cell culture SKOV3 and SKOV3/DDP cells were purchased from Shanghai Fuyu Biotechnology Co., LTD. McCoy '5a (Tianhang Bio, Hangzhou, China) with 10% Fetal Bovine Serum (FBS) (Service Bio, Wuhan, China)was cultured in an incubator with 37 ℃ and 5% CO 2 . In addition, SKOV3/DDP cells need to be added 0.5ug/ml DDP (Meilun Bio Dalian, China) to maintain DDP resistance. Cell viability and combination index (CI) The SKOV3 and SKOV3/DDP cells of the DDP group were subjected to varying concentrations of cisplatin (0 µg/mL, 1.5 µg/mL, 3 µg/mL, 6 µg/mL, 12 µg/mL, 24 µg/mL) and cultured in a 37℃, 5% CO 2 incubator. Meanwhile, for the ASP group, SKOV3 and SKOV3/DDP cells were treated with different concentrations of ASP (Batch Number: C23A6Y1, Yuanye Bio, Shanghai, China) (0 µg/mL, 50 µg/mL, 100 µg/mL, 150 µg/mL, 200 µg/mL, 250 µg/mL, 300 µg/mL) and also cultured in a 37℃, 5% CO 2 incubator for 48 hours. In the ASP combined with DDP group, SKOV3/DDP cells were subjected to 10 µg/mL DDP concentration, followed by the addition of different concentrations (0 µg/mL, 50 µg/mL, 100 µg/mL, 150 µg/mL, 200 µg/mL, 250 µg/mL, 300 µg/mL) of ASP in each group. The cell viability assay was conducted using CCK8 (Wanlei Bio, Shenyang, China). Furthermore, we used CompuSyn software (CompuSyn Inc.) to determine the Combination index (CI). The study results indicated the following: CI = 0.85 to 0.9 representing slight synergism; CI = 0.7 to 0.85 representing moderate synergism; CI = 0.3 to 0.7 representing synergism; CI = 0.1 to 0.3 representing strong synergism; and CI < 0.1 representing very strong synergism. In vivo xenograft assays The study utilized female BALB/c nude mice obtained from Cavens Laboratory Animal Co., Ltd. The mice, which were 4 weeks old, were split into ten mice per group. To induce tumor formation, SKOV3/DDP cells were dissolved in 20 µL of phosphate-buffered saline (PBS) and then subcutaneously injected into the armpits of the mice. The growth of the tumors to 100 mm 3 was set as day 1. For the Vehicle (Veh) group, the nude mice were intraperitoneally injected with 50 mL/kg/d PBS. Meanwhile, the DDP group was intraperitoneally injected with 4 mg/kg DDP on day 1 and 8 [ 15 ]. Following Hu Zhuang et al.'s research on restraining breast tumor growth through the intraperitoneal injection of 0.2 mg/kg/d ASP in nude mice [ 16 ], the ASP group mice were also intraperitoneally injected with 0.2 mg/kg/d ASP. The tumors of the mice in each group were monitored every three days to determine the tumor volume ( Fig. 3 A ) .After 21 days, the mice were euthanized, and the tumors were collected for analysis ( Fig. 3 B ) . The animal experiment was conducted following the Guidelines for the Care and Use of Laboratory Animals and approved by the Ethics Committee of Harbin Medical University (certificate number: KY2023-37).. Histological analysis We took tissue extraction and embedding sections from the liver, kidney, and heart of the killed nude mice. Hematoxylin (Solarbio, Shanghai, China) and eosin Y (Sangon, Shanghai, China) were used for staining. Histological sections were taken with a microscope (OLYMPUS, Japan). Cell invasion assays The Transwells (Labselect, Hefei, China) were mixed with Matrigel (Corning, USA) and placed into a 24-well plate, which was then placed in a 37℃ incubator for 2 hours to allow the Matrigel to solidify. Next, 5×10 4 cells per well were added to the upper chamber and 800 µl of culture medium, containing 10% FBS, was added to the lower chamber. After 24 hours, the transwells were removed, washed twice with PBS, fixed with 4% neutral paraformaldehyde (Aladdin, Shanghai, China) for 20 minutes, and stained with 0.1% crystal violet (Amresco, USA) for 5 minutes. Finally, cells on the outer surface of the membrane were observed with a microscope and the number of cells passing through the permeable membrane in the visual field was randomly counted. RNA-seq analysis SKOV3/DDP cells were treated with either DDP or ASP combined with DDP. RNA was then extracted from both groups of cells using TRIzol (TaKaRa Bio, Dalian, China). The experiment was repeated three times using independent biological samples. The high-throughput sequencing platform NovaSeq 6000 by Illumina was utilized to sequence the samples. Raw data were filtered and assessed for quality. The clean reads were mapped to the human genome (GENCODE download, GRCh38) for Inter-sample expression level and principal component analysis. The DESeq2 R package was employed to analyze the raw data, and the differential expression threshold for DEGs was set to P value 1. DEGs were subjected to GO and KEGG analyses using David (v2022q2, https://david-d.ncifcrf.gov/ ). Lentivirus infection The sequence of GPX4 gene (NM_001039848.4, GENERAL Bio, Chuzhou, China) was linked to the pLVX-IRES-puro vector (Fenghui Bio, Changsha, China) and transfected into 293T cells. After 48 hours, the lentivirus supernatant was collected and used to transfect SKOV3/DDP cells for 12 hours. Subsequently, the cells were screened with puromycin for 7 days to select for GPX4 overexpression. The overexpression of GPX4 was confirmed using RT-qPCR and Western blotting. RT-qPCR RNA extracted from SKOV3/DDP cells in each group was obtained via TRIzol (TaKaRa Bio, Dalian, China). Then, mRNA was reverse-transcribed into cDNA with the BeyoRT™ II cDNA First Strand Synthesis Kit (Beyotime, Shanghai, China). GAPDH was used as the internal parameter. The reaction system consisted of 1 µL cDNA and 10 µL SYBR Green Master Mix (Solarbio, Beijing, China), 0.5 µL of upstream and downstream primers each (GENERAL Bio, Chuzhou, China), and 8 µL ddH 2 O. The reaction conditions included an initial denaturation of 95°C for 5 min, denaturation of 95°C for 10 s, annealing of 60°C for 10 s, extension of 72°C for 1 min (40 cycles), and final extension of 1 min 30s at 72°C. The primer sequences used were GPX4 F, GAAGCAGGAGCCAGGGAGT and GPX4 R, CGCAGCCGTTCTTGTCG; β-actin F, GGCACCCAGCACAATGAA and β-actin R, TAGAAGCATTTGCGGTGG. Western blot Each group was lysed with cell lysis solution (Wanlei Bio, Shenyang, China). The BCA protein concentration assay kit (Wanleibio, Shenyang, China) was used to quantify the protein concentration of each group of cells. The proteins were isolated by 14% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Wanlei Bio, Shenyang, China) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). PVDF membrane was sealed with 5% skim milk for 1 hour and incubated overnight with primary anti-GPX4 (Abclonal, Wuhan, China) and anti-β-actin (Wanlei Bio, Shenyang, China) at 4℃. Sheep Anti-Rabbit IgG-HRP (Wanlei Bio, Shenyang, China) was added and incubated at 37℃ for 45min. Finally, ECL (Wanlei Bio, Shenyang, China) was added for exposure scanning, and Gel-Pro-Analyzer software was used to analyze the optical density of the strips. Ferroptosis determination Fe 2+ , MDA, SOD, and GSH are indicators of ferroptosis. To measure the contents of these indicators, we used Ferrous Iron Colorimetric Assay Kit (Elabscience, Wuhan, China), MDA Colorimetric Assay Kit (Wanlei Bio, Shenyang, China), Total Superoxide Dismutase Activity Assay Kit (Wanlei Bio, Shenyang, China), and Reduced Glutathione Colorimetric Assay Kit (Wanlei Bio, Shenyang, China) following the manufacturer's instructions. We measured the absorbance and calculated the concentrations of Fe 2+ , MDA, SOD, and GSH in each cell group. Statistical analysis We used GraphPad Prism 8.0 software to analyze the results of the experiments. The student t-test was used to compare the differences between two groups, while ANOVA was used to compare between three or more groups. Statistical significance was set at P < 0.05. Results ASP and DDP can inhibit the proliferation of SKOV3 and SKOV3/DDP SKOV3 and SKOV3/DDP cells were treated with DDP (1.5µg/ml, 3 µg/ml, 6µg/ml, 12µg/ml, 24 µg/ml), and cell viability was measured 48 hours later. The results are shown below, SKOV3 groups: compared with the vehicle group, the viability of each group was 81.95%±5.37, 57.70%±7.66, 43.28%±7.09, 35.22%±4.07, 24.56%±3.03; SKOV3/DDP groups: Compared with the vehicle group, the viability of each group was 94.07%±4.36, 88.15%±10.89, 79.20%±9.10, 66.82%±7.92, 53.08%±7.58 ( Fig. 1A ) . The viability of each group decreased with increasing DDP concentration when compared to the vehicle group. Similarly, SKOV3/DDP groups showed a decrease in viability with increasing DDP concentration. Student t-test was used to compare the proliferative activity of DDP against SKOV3 and SKOV3/DDP cells, showing significant differences in sensitivity to DDP (P<0.01). We also treated SKOV3 and SKOV3/DDP cells with ASP (50 µg/mL, 100 µg/mL, 150 µg/mL, 200 µg/mL, 250 µg/mL, 300µg/mL), and cell viability was measured 48 hours later. SKOV3 group: Compared with the vehicle group, the viability of each group was 98.95%±8.59, 97.03%±7.57, 94.96%±13.33, 90.37%±6.49, 82.54%±3.83, 76.33%±3.56. SKOV3/DDP group: Compared with the vehicle group, the viability of each group was 98.76%±8.13, 97.61%±6.73, 96.37%±6.56, 90.72%±5.05, 87.86%±0.64, 83.92%±1.41 ( Fig. 1B ) .Both SKOV3 and SKOV3/DDP groups showed a decrease in viability with increasing ASP concentration. Overall, ASP inhibited the proliferation of SKOV3 and SKOV3/DDP cells, with increasing inhibition at higher concentrations of ASP. ASP combined with DDP increases the sensitivity of SKOV3/DDP to DDP To assess the efficacy of the combination of ASP and DDP on SKOV3/DDP cells, we calculated the combination index (CI) of ASP and DDP. Each group was supplemented with 10 µg/mL of DDP and varying concentrations of ASP. After 48 hours, cell viability was examined and compared to that of the control group. Cell viability decreased with increasing concentrations of ASP, with values of 75.74%±6.76, 70.39%±6.77, 59.96%±8.83, 56.90%±2.91, 51.95%±3.88, 47.47%±3.93, and 38.33%±4.87% ( Fig. 2A ) . Using CompuSyn software, we calculated the CI for each group and obtained values of 1.06, 0.73, 0.71, 0.65, 0.60, and 0.47 ( Fig. 2B ) . These results indicate that ASP and DDP exhibit a synergistic effect, except for ASP at a concentration of 50 µg/mL. Effect of ASP combined with DDP on SKOV3/DDP in vivo, and the evaluation of safety The tumor size and growth rate in nude mice were lower in the ASP combined with DDP group than in the other groups. ASP combined with DDP showed higher tumor inhibition rates (52.05%, 48.93%, and 73.99%) than ASP group, DDP group, and vehicle group ( Fig. 3C ) . We collected liver, kidney, and heart tissue from the mice and used HE staining to assess the safety of ASP and DDP treatment in vivo. The results indicated that ASP combined with DDP had no significant effect on the morphology of the liver, kidney, or heart, suggesting that it is a safe treatment option ( Fig. 3D ) . Effects of ASP combined with DDP on SKOV3/DDP cell invasion The effects of the ASP group, the DDP group, and ASP combined with DDP group on SKOV3/DDP cell invasion were investigated using the Transwell method. The combined ASP and DDP group demonstrated lower average cell counts passing through the permeable membrane of the Transwell compared to the ASP and DDP groups. The ASP combined with DDP showed a stronger inhibition of SKOV3/DDP cell invasion ( Fig. 4A &B) . The cell's transcriptome was sequenced to identify key genes To investigate whether the combination of ASP and DDP can regulate key genes in SKOV3/DDP transcription process, we conducted transcriptome sequencing on two groups: ASP combined with DDP and DDP alone. First, the correlation analysis ( Fig. 5A ) and principal component analysis (PCA) ( Fig. 5B ) indicate that the obtained data is reliable and there are significant differences between the data of the DDP group and the ASP combined with DDP group. Next, using the defined threshold, we obtained 843 DEGs ( Fig. 5C ) , among which 405 down-regulated genes and 438 up-regulated genes existed ( Fig. 5D ) (Table.1s) .We then analyzed these DEGs by GO and KEGG. The GO enrichment analysis showed that Molecular Function (MF) was mainly enriched in structural constituent of chromatin, structural constituent of ribosome, and protein heterodimerization activity. Biological Processes (BP) were mainly concentrated in cytoplasmic translation, nucleosome assembly, and translation. Cellular Component (CC) was mainly concentrated in nucleosome and mitochondrial inner membrane ( Fig. 6A &B) (Table.2s) . KEGG enrichment analysis showed that DEGs were mainly concentrated in oxidative phosphorylation, tumor transcriptional regulation, and other pathways ( Fig. 6C &D) (Table.3s) . Previous studies by Xiaodong Wu et al. have found that the expression level of GPX4 in SKOV3/DDP cells is higher than in SKOV3 cells and that its increase is associated with the prognosis and drug resistance of patients[17]. After conducting a comprehensive analysis on both log 2 Fold Change ranking and P-value ranking, as well as reviewing relevant publications, it is postulated that GPX4 is a key gene involved in the regulation of SKOV3/DDP cells. Consequently, our subsequent experiment will be centered on the exploration of GPX4. RTqPCR and western blot analysis of key genes GPX4 We treated SKOV3/DDP cells with ASP, DDP, and a combination of ASP and DDP. GPX4 expression was detected via both RT-qPCR ( Fig. 7A ) and western blot ( Fig. 7B &C) . Our results showed that GPX4 was significantly down-regulated in the group treated with a combination of ASP and DDP. Furthermore, to investigate the role of GPX4 in SKVO3/DDP cells, we generated stable SKVO3/DDP cells that overexpress GPX4, referred to as SKOV3/DDP-GPX4 cells. We then compared the expression of GPX4 in SKOV3/DDP, SKOV3/DDP-NC (negative control group), and SKOV3/DDP-GPX4 cells using RT-qPCR ( Fig. 7D ) and western blot ( Fig. 7E &F) . The data suggested that GPX4 was highly expressed in SKOV3/DDP-GPX4 cells. ASP combined with DDP inhibited SKOV3/DDP proliferation and invasion by inhibiting GPX4 The control group, SKOV3/DDP-NC group, and SKOV3/DDP-GPX4 group were treated with Veh, DDP, ASP combined with DDP, respectively, and the cell proliferation of each group was detected using CCK8. The results showed that the cell viability of the control group, SKOV3/DDP-NC group, and SKOV3/DDP-GPX4 group treated with ASP combined with DDP was significantly lower than that of the group treated with DDP alone (Fig. 8A) . Similarly, transwell assay was performed to assess the cell invasion ability, and it was observed that the cell invasion ability of the control group, SKOV3/DDP-NC group, and SKOV3/DDP-GPX4 group treated with ASP combined with DDP was significantly lower than that of the group treated with DDP alone (Fig. 8B&C) . These results indicate that ASP combined with DDP can effectively inhibit cell proliferation and invasion by suppressing GPX4. ASP combined with DDP can cause ferroptosis of SKOV3/DDP by inhibiting GPX4 expression of SKOV3/DDP To investigate how ASP combined with DDP facilitates ferroptosis in SKOV3/DDP cells, we conducted experiments to determine the levels of Fe 2+ , MDA, SOD, and GSH. The control group, SKOV3/DDP-NC group, and SKOV3/DDP-GPX4 group were treated with Veh, DDP, and ASP combined with DDP. We observed a significant increase in Fe 2+ and MDA content in all groups following the treatment of ASP combined with DDP ( Fig. 9A &B) . Conversely, the levels of SOD and GSH were significantly reduced in all groups treated with ASP combined with DDP ( Fig. 9C &D) . Discussion At present, drug resistance remains a significant cause of poor prognosis in individuals with cancer[ 18 ]. Research has indicated that drug resistance in tumors is connected to the tumor microenvironment, and traditional Chinese medicine may be able to reverse drug resistance by regulating the tumor microenvironment [ 19 ]. ASP possesses multiple functions, such as anti-tumor capabilities, the regulation of cell microenvironment, and the treatment of gynecological diseases[ 6 , 20 ]. Thus, we hypothesize that ASP can reverse the drug resistance of ovarian cancer by regulating its microenvironment or substance metabolism. Initially, we conducted a verification experiment on SKOV3 and the sensitivity of SKOV3/DDP cells to DDP. The experiment demonstrated that the sensitivity of SKOV3/DDP cells to DDP was lower than that of SKOV3 cells. We also conducted experiments on whether ASP has an inhibitory effect on SKOV3 and SKOV3/DDP cells. The results indicated that ASP had an inhibitory effect on SKOV3 but had a poor inhibitory effect on SKOV3/DDP cells. To better understand whether the combination of two drugs has a synergistic effect, we calculated the combination index (CI) values of the two drugs[ 21 ]. The results showed that the two drugs had excellent synergistic effects. Additionally, we performed in vivo testing with the drugs. After subcutaneously forming tumors in nude mice, we intraperitoneally injected the drugs and periodically measured the condition of the tumors in the mice. The results showed that the combination of ASP and DDP had an excellent tumor inhibition effect. Furthermore, evaluating drug safety is crucial when considering drug clinical applications [ 22 ]. It has been reported that ASP has antioxidant protection on cardiac cells [ 23 ] and reduce liver fibrosis induced by ccl 4 [ 24 ].The liver, heart, and kidney conditions were evaluated in each group of nude mice, and no differences were found among them, demonstrating that ASP combined with DDP had good safety. Tumor invasion is related to drug resistance [ 25 ], so the effects of ASP, DDP, and ASP combined with DDP on SKOV3/DDP invasion were investigated. The results revealed that ASP combined with DDP could better inhibit SKOV3/DDP invasion. Next, we explored the specific mechanism by which ASP combined with DDP changes the resistance of SKOV3/DDP cells to DDP. Comparison of the RNA-seq data between the DDP group and the ASP combined with DDP group revealed the presence of numerous DEGs involved in transcription. GO [ 26 ] and KEGG[ 27 ] analyses of the DEGs showed enrichment in metabolic processes and cellular transcriptional regulation. Specifically, cancer cells undergo different metabolic processes from normal cells[ 28 ], with increased glycolysis demonstrated by the Warburg effect [ 29 ] and a dependence on rapid tricarboxylic acid (TCA) cycling to sustain proliferation [ 30 ]. The Warburg effect has also been linked to drug resistance [ 31 ]. Additionally, drug resistance in ovarian cancer has been associated with cellular transcriptional regulation [ 32 ]. In conclusion, the analysis suggests a relationship between the combination of ASP with DDP and the regulation of drug resistance in SKOV3/DDP cells. We sequenced differentially expressed genes based on their log 2 fold change and p-value, and reviewed the relevant literature. According to previous studies, GPX4 expression is higher in SKOV3/DDP cells compared to SKOV3 cells, and inhibition of GPX4 can reduce drug resistance in resistant cells[ 17 ]. Therefore, we hypothesized that downregulation of GPX4, caused by the combination of ASP and DDP, is responsible for the decreased resistance of SKOV3/DDP cells. In subsequent experiments, SKOV3/DDP cells were stably transfected with lentivirus to overexpress GPX4, and relevant experiments were conducted. The results showed that overexpressing GPX4 in SKOV3/DDP cells increased their resistance to DDP, while combining ASP with DDP reversed DDP resistance. Studies have shown that GPX4 is a crucial gene involved in regulating ferroptosis in cells [ 33 ]. Ferroptosis is a complex biological process characterized by a toxic accumulation of lipid peroxides on cell membranes[ 34 , 35 ]. It has also been associated with tumor occurrence and development [ 36 ]. Specifically, Wang Y et al. demonstrated that the regulation of ferroptosis can affect the sensitivity of platinum-resistant ovarian cells to DDP[ 37 ]. The presence of Fe 2+ [ 38 ]and MDA[ 39 ], which are products of membrane lipid peroxidation, plays a crucial role in detecting ferroptosis. Moreover, SOD [ 40 ] and GSH [ 41 ] are also used as diagnostic criteria for ferroptosis in cells. Experimental results revealed that the combination of ASP and DDP treatment led to a significant increase in Fe 2+ and MDA levels, while SOD and GSH levels were significantly decreased. These changes indirectly indicate a reduction in GPX4 expression and an exacerbation of ferroptosis in SKOV3/DDP cells. ( Fig. 10 ) . Because ASP is predominantly composed of glucose (Glc), galactose (Gal), arabinose (Ara), rhamnose (Rha), fucose (Fuc), xylose (Xyl), and galacturonic acid (GalUA)[ 6 ], we will conduct further investigations on the monosaccharide components in ASP in order to examine their impact on tumor. Additionally, our study reveals an interesting finding from the GO analysis, indicating an enrichment of the Cellular Component (CC) in the mitochondrial inner membrane. It is well-known that cellular ferroptosis induces alterations in mitochondria [ 42 ]. Mitochondria, being instrumental in cysteine-deprivation induced (CDI) ferroptosis, do not, however, play a significant role in inhibiting GPX4-induced ferroptosis[ 43 ]. As a result, we question whether ASP combined with DDP possesses an alternative mechanism for regulating the ferroptosis of SKOV3/DDP cells, aside from its influence on GPX4. Moreover, it is worth noting that GPX4 expression is frequently controlled by Non-coding RNA [ 44 ]. We hypothesize that the combination of ASP and DDP can regulate the GPX4 expression in SKOV3/DDP through non-coding RNA, thereby promoting an increase in SKOV/DDP ferroptosis. This aspect will also be one of our focal points of investigation. Due to the intricate mechanism of ferroptosis in tumors, additional experiments will be conducted to determine the specific mechanism by which ASP combined with DDP inhibits GPX4 expression. Conclusions We discovered that ASP combined with DDP can regulate the transcription process of SKOV3/DDP, and it can inhibit the expression of the key gene GPX4 to promote the ferroptosis of SKOV3/DDP. This finding provides a theoretical basis for the clinical application of ASP. Abbreviations ASP Angelica Sinensis Polysaccharide DDP cisplatin MDA malondialdehyde SOD superoxide dismutase GSH Glutathione HE Hematoxylin-eosin GPX4 Recombinant Glutathione Peroxidase 4 RNA-seq RNA-sequencing DEGs Differentially Expressed Genes CCK8 Cell Counting Kit-8 PBS Phosphate Buffered Saline FBS Fetal Bovine Serum CI Combination Index PVDF polyvinylidene fluoride Veh Vehicle group GO Gene Ontology KEGG Kyoto Encyclopedia of Genes and Genomes MF Molecular Function BP Biological Processes CC Cellular Component MDA Malondialdehyde CDI cysteine-deprivation induced Declarations Acknowledgements Not applicable. Author Contributions Experiment Design: Yaoxian Wang Weikang Guo. Experiment Operation: Weikang Guo, Fei Lei, Ruxin Zheng, Xinyao Zhao,Yunshun Tong.Data Statistics: Yuze Gu, Mengdi Yang, Yunshun Tong. Article Written: Weikang Guo,Wanyue Wang. Disclosure statement The authors declare no competing interests. Funding WU JIEPING Medical Foundation Special Fund for Clinical Research (No.320.6750.2023-05-4). Data availability All datasets generated for this study are included in the article/ supplementary material, further inquiries can be directed to the corresponding authors. Ethics approval and consent to participate The animal study was reviewed and approved by the Ethics Committee of Harbin Medical University(certificate number: KY2023-37). Consent for publication Manuscript is approved by all authors for publication. Competing interests The authors declare no competing interests. Author details 1Department of Gynecology, Harbin Medical University Cancer Hospital, Harbin,150081, China 2. School of Basic Medical Sciences, Qiqihar Medical University, Qiqihar,161006,China 3. 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Wang K, Wang J, Song M, Wang H, Xia N, Zhang Y: Angelica sinensis polysaccharide attenuates CCl(4)-induced liver fibrosis via the IL-22/STAT3 pathway. Int J Biol Macromol 2020, 162:273-283. Liang Y, McDonnell S, Clynes M: Examining the relationship between cancer invasion/metastasis and drug resistance. Curr Cancer Drug Targets 2002, 2(3):257-277. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT et al: Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 2000, 25(1):25-29. Kanehisa M, Goto S: KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 2000, 28(1):27-30. Xiong Y, Wang L, Feng Y, Li X, Jia L, Han Y: Flexibility in metabolism bestows tenacious viability on cancer. Life Sci 2018, 208:20-25. Liu C, Jin Y, Fan Z: The Mechanism of Warburg Effect-Induced Chemoresistance in Cancer. Front Oncol 2021, 11:698023. Eniafe J, Jiang S: The functional roles of TCA cycle metabolites in cancer. Oncogene 2021, 40(19):3351-3363. Bhattacharya B, Mohd Omar MF, Soong R: The Warburg effect and drug resistance. Br J Pharmacol 2016, 173(6):970-979. Richardson A, Kaye SB: Drug resistance in ovarian cancer: the emerging importance of gene transcription and spatio-temporal regulation of resistance. Drug Resist Updat 2005, 8(5):311-321. Seibt TM, Proneth B, Conrad M: Role of GPX4 in ferroptosis and its pharmacological implication. Free Radic Biol Med 2019, 133:144-152. Hassannia B, Vandenabeele P, Vanden Berghe T: Targeting Ferroptosis to Iron Out Cancer. Cancer Cell 2019, 35(6):830-849. Mou Y, Wang J, Wu J, He D, Zhang C, Duan C, Li B: Ferroptosis, a new form of cell death: opportunities and challenges in cancer. J Hematol Oncol 2019, 12(1):34. Lei G, Zhuang L, Gan B: Targeting ferroptosis as a vulnerability in cancer. Nat Rev Cancer 2022, 22(7):381-396. Wang Y, Zhao G, Condello S, Huang H, Cardenas H, Tanner EJ, Wei J, Ji Y, Li J, Tan Y et al: Frizzled-7 Identifies Platinum-Tolerant Ovarian Cancer Cells Susceptible to Ferroptosis. Cancer Res 2021, 81(2):384-399. Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, Patel DN, Bauer AJ, Cantley AM, Yang WS et al: Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 2012, 149(5):1060-1072. Ursini F, Bosello Travain V, Cozza G, Miotto G, Roveri A, Toppo S, Maiorino M: A white paper on Phospholipid Hydroperoxide Glutathione Peroxidase (GPx4) forty years later. Free Radic Biol Med 2022, 188:117-133. Song JX, An JR, Chen Q, Yang XY, Jia CL, Xu S, Zhao YS, Ji ES: Liraglutide attenuates hepatic iron levels and ferroptosis in db/db mice. Bioengineered 2022, 13(4):8334-8348. Ursini F, Maiorino M: Lipid peroxidation and ferroptosis: The role of GSH and GPx4. Free Radic Biol Med 2020, 152:175-185. Wang H, Liu C, Zhao Y, Gao G: Mitochondria regulation in ferroptosis. Eur J Cell Biol 2020, 99(1):151058. Fernandez-Acosta R, Hassannia B, Caroen J, Wiernicki B, Alvarez-Alminaque D, Verstraeten B, Van der Eycken J, Vandenabeele P, Vanden Berghe T, Pardo-Andreu GL: Molecular Mechanisms of Nemorosone-Induced Ferroptosis in Cancer Cells. Cells 2023, 12(5). He GN, Bao NR, Wang S, Xi M, Zhang TH, Chen FS: Ketamine Induces Ferroptosis of Liver Cancer Cells by Targeting lncRNA PVT1/miR-214-3p/GPX4. Drug Des Devel Ther 2021, 15:3965-3978. Additional Declarations No competing interests reported. Supplementary Files Table1s.xlsx Supplementary Information Additional fle : Table S1. DEGs from DDP group and the ASP combined with DDP group transcriptional sequencing. Table2s.xlsx Table S2. The result of GO analysis Table3s.xlsx Table S2. The result of GO analysis Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3312243","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":230125613,"identity":"413a8569-52bd-4545-85a7-c35b83c6cbeb","order_by":0,"name":"Weikang Guo","email":"","orcid":"","institution":"Harbin Medical University Cancer Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Weikang","middleName":"","lastName":"Guo","suffix":""},{"id":230125614,"identity":"0d8bcb8b-9e15-480e-8eab-e01e8c357dfc","order_by":1,"name":"Wanyue Wang","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wanyue","middleName":"","lastName":"Wang","suffix":""},{"id":230125615,"identity":"87664434-2e47-4e24-b419-181b7bc76ad0","order_by":2,"name":"Fei Lei","email":"","orcid":"","institution":"Harbin Medical University Cancer Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Lei","suffix":""},{"id":230125616,"identity":"351847c4-8b94-49ef-afd3-7cd6e1712b20","order_by":3,"name":"Ruxin Zheng","email":"","orcid":"","institution":"Harbin Medical University Cancer Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ruxin","middleName":"","lastName":"Zheng","suffix":""},{"id":230125617,"identity":"5281eea5-16da-4224-a120-3785f45eb78d","order_by":4,"name":"Xinyao Zhao","email":"","orcid":"","institution":"Harbin Medical University Cancer 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05:44:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3312243/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3312243/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":42678656,"identity":"e55d0c6c-f6ca-4bb3-97b2-4a9a07ccde5f","added_by":"auto","created_at":"2023-09-06 01:16:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":320540,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eASP and DDP can inhibit the proliferation of SKOV3 and SKOV3/DDP A. \u003c/strong\u003eInfluence of different DDP concentrations on SKOV3 and SKOV3/DDP at 48 h. \u003cstrong\u003eB. \u003c/strong\u003eInfluence of different ASP concentrations on SKOV3 and SKOV3/DDP at 48 h.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3312243/v1/1100b5b57ca43dcd0fb286e0.jpg"},{"id":42679719,"identity":"f020c8fa-5897-4d28-a33c-42e77ded040a","added_by":"auto","created_at":"2023-09-06 01:24:55","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":304429,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eASP collaboration with DDP can inhibit SKOV/DDP proliferation\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003eQuantitative DDP and different concentrations of ASP were added to SKOV3/DDP cells to show cell proliferation in each group at 48 hours \u003cstrong\u003eB.\u003c/strong\u003eCI values of ASP of different concentrations combined with DDP\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3312243/v1/ea84def5f7e3f5e2c8bb7fbf.jpg"},{"id":42678665,"identity":"e90350af-c10c-49fd-b63d-abbe0888ce1f","added_by":"auto","created_at":"2023-09-06 01:16:55","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9834604,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eASP combined with DDP can inhibit the growth of SKOV3/DDP in vivo, and has certain safety. A.\u003c/strong\u003eSpecific ways of ASP combined with DDP treatment in nude mice \u003cstrong\u003eB.\u003c/strong\u003eTumor size after death of nude mice in each group \u003cstrong\u003eC. \u003c/strong\u003eThe tumor size of nude mice in each group was measured every 3 days \u003cstrong\u003eD.\u003c/strong\u003eHE stained sections of liver, kidney and heart of nude mice in each group were observed and compared under the microscope (scale was 100μm).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3312243/v1/97df864da2a48c8c6221f5c0.jpg"},{"id":42678658,"identity":"37be6fe6-7675-4d82-bf24-429f93f3eb8b","added_by":"auto","created_at":"2023-09-06 01:16:55","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":486453,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eASP combined with DDP can affect the invasion ability of SKOV3/DDP A.\u003c/strong\u003e The effects of ASP, DDP, ASP combined with DDP on the invasion ability of SKOV3/DDP cells were detected by Transwell. (200×) \u003cstrong\u003eB.\u003c/strong\u003eThe number of cells on the lateral surface of the membrane was observed under the microscope.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3312243/v1/43231ddd3e849a93b8fea78c.jpg"},{"id":42678663,"identity":"66299a62-6285-4c3b-8da1-3c28cda929ae","added_by":"auto","created_at":"2023-09-06 01:16:55","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1022800,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential gene analysis of DDP group and ASP combined with DDP during transcription.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003e Correlation heat map between pairs based on expression abundance. The closer the value in the box is to 1, the more similar it is. (C1, C2, and C3 belong to DDP group and D1, D2, and D3 belong to ASP combined with DDP group) \u003cstrong\u003eB. \u003c/strong\u003ePrincipal component analysis data.\u003cstrong\u003e C. \u003c/strong\u003eHeat map. The more red, the more up-regulated the gene expression, and the more blue, the more down-regulated the gene expression. \u003cstrong\u003eD. \u003c/strong\u003eDifferential gene volcano map. Blue represents down-regulated genes, red up-regulated genes, and gray represents genes with no difference in expression.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3312243/v1/4c56d329d312f713cf311b7e.jpg"},{"id":42679717,"identity":"422f3d10-1c94-4cb6-b4b9-3de7c5eb6cae","added_by":"auto","created_at":"2023-09-06 01:24:55","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1064589,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential gene GO and KEGG analysis. A\u0026amp;C.\u003c/strong\u003e The X-axis represents the p value enriched in GO_term or KEGG_pathway; the Y-axis represents a certain GO_term or KEGG_pathway; the color represents different classifications; the number represents the number of genes enriched in this term or pathway. \u003cstrong\u003eB\u0026amp;D. \u003c/strong\u003eThe X-axis represents the ratio of the number of genes in this term or pathway in the differentially expressed genes to the total number of genes in this term or pathway in all annotated genes.The size of the circle represents the number of DEGs enriched on the GO_term or KEGG_pathway.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3312243/v1/e9361b16209dce6eb4019cd5.jpg"},{"id":42678660,"identity":"7f6f89a3-fae4-4400-a7e2-b56acb1f550a","added_by":"auto","created_at":"2023-09-06 01:16:55","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":424080,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eASP combined with DDP inhibited GPX4 expression of SKOV3/DDP, and SKOV3/DDP overexpressing GPX4 was constructed. A.\u003c/strong\u003emRNA expression levels of GPX4 in ASP group, DDP group and ASP combined DDP group. \u003cstrong\u003eB\u0026amp;C. \u003c/strong\u003eGPX4 protein expression in each group.β-actin is the reference protein. \u003cstrong\u003eD. \u003c/strong\u003emRNA expression levels of GPX4 in each cell group. \u003cstrong\u003eE\u0026amp;F.\u003c/strong\u003e GPX4 protein expression in each cell group.β-actin is the reference protein.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3312243/v1/734251b011fe97058324e3cf.jpg"},{"id":42679720,"identity":"e6b3c619-0afe-4daa-9a02-f11537899b4e","added_by":"auto","created_at":"2023-09-06 01:24:55","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":938725,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eASP combined with DDP inhibited SKOV3/DDP proliferation and invasion by inhibiting GPX4\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003eThe effects of Veh, DDP, and ASP combined with DDP on cell proliferation were detected at 48 h. \u003cstrong\u003eB.\u003c/strong\u003e Effects of Veh, DDP, and ASP combined with DDP on cell invasion in each group. (200×) \u003cstrong\u003eC.\u003c/strong\u003eThe number of cell invasions in each group under the microscope.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3312243/v1/a9f05f7aafa2c5491cdfb5c9.jpg"},{"id":42678664,"identity":"dabdc6fb-a89a-4bf2-8129-5b8371e90bb8","added_by":"auto","created_at":"2023-09-06 01:16:55","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":3438139,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eASP combined with DDP can promote \u003c/strong\u003e\u003ca href=\"javascript:;\"\u003e\u003cstrong\u003eferroptosis\u003c/strong\u003e\u003c/a\u003e\u003cstrong\u003e of SKOV3/DDP by inhibiting GPX4. \u003c/strong\u003eThe content of Fe\u003csup\u003e2+\u003c/sup\u003e, MDA, SOD, and GSH in each group of cells can be expressed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Fe\u003csup\u003e2+\u003c/sup\u003e content. \u003cstrong\u003eB.\u003c/strong\u003e MDA content. \u003cstrong\u003eC.\u003c/strong\u003e SOD content. \u003cstrong\u003eD.\u003c/strong\u003e GSH content.\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3312243/v1/2bd77f096349c350255b914a.jpg"},{"id":42678669,"identity":"b22760ea-7243-4a48-8a7e-8e83e88d1495","added_by":"auto","created_at":"2023-09-06 01:16:55","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":255126,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanism diagram of ASP combined with DDP enhancing ferroptosis by inhibiting GPX4 expression of SKOV3/DDP.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3312243/v1/c4a2a59a6005ac72ce889c93.jpg"},{"id":44049489,"identity":"c775981b-729b-48fb-885c-0331a5a3f0d3","added_by":"auto","created_at":"2023-10-04 00:37:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1518041,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3312243/v1/f4dbaec3-6a49-480d-8074-5da39595511d.pdf"},{"id":42678667,"identity":"7c5f7d32-f073-4943-9a77-79c3a02a9a3c","added_by":"auto","created_at":"2023-09-06 01:16:55","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":166411,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional fle : Table S1. DEGs from DDP group and the ASP combined with DDP group transcriptional sequencing.\u003c/p\u003e","description":"","filename":"Table1s.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3312243/v1/24987cde0dc7b9bd0f3c45f3.xlsx"},{"id":42679718,"identity":"95e72539-ba60-4384-a387-000a32c6e3b0","added_by":"auto","created_at":"2023-09-06 01:24:55","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":39956,"visible":true,"origin":"","legend":"\u003cp\u003eTable S2. The result of GO analysis\u003c/p\u003e","description":"","filename":"Table2s.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3312243/v1/9f1b24d1f9b34996d26e8b01.xlsx"},{"id":42678657,"identity":"da287219-d0a7-48a4-907a-ed035c4d701d","added_by":"auto","created_at":"2023-09-06 01:16:55","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":40716,"visible":true,"origin":"","legend":"\u003cp\u003eTable S2. The result of GO analysis\u003c/p\u003e","description":"","filename":"Table3s.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3312243/v1/d28c7ebc5a43df39b08b4f89.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The combination of Angelica sinensis polysaccharide and cisplatin promotes ferroptosis in cisplatin-resistant ovarian cancer cells by regulating GPX4, thereby reversing their resistance to cisplatin","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOvarian cancer is among the top three malignancies affecting women[1]. Ovarian cancer is often diagnosed at a later stage due to the absence of typical early symptoms[2]. Cytoreductive surgery and postoperative platinum-based chemotherapy remain the standard treatments for ovarian cancer[1]. While medical technology has advanced and initial treatment outcomes for ovarian cancer have improved, many patients eventually develop chemotherapy resistance, leading to tumor recurrence and metastasis. Cisplatin (DDP) is the representative drug that patients tend to develop resistance to[3]. Hence, investigating the signaling pathways and targeted drugs tied to multi-drug resistance in ovarian cancer and, subsequently, addressing the phenomenon of multi-drug resistance, is critical for enhancing patient survival rates[4]. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAngelica sinensis (Oliv.) Diels is a classic Chinese medicine prescription that has been utilized for the treatment of various gynecological diseases for thousands of years in China[5]. Angelica sinensis polysaccharide (ASP) is a major extracted component of Angelica sinensis[6]. ASP enhances the immune function of the body and acts as an immunomodulator[7], and possesses antioxidant abilities that help protect the liver[8], heart[9], and kidney[10]. Recent studies have found that ASP exhibits an antitumor effect and may aid in treating lung, liver, breast[11], and cervical cancers[12].In addition, in order to make ASP have a higher therapeutic effect on tumors, researchers analyzed ASP using 1H-NMR\u0026nbsp;and FT-IR spectroscopy and created a hypoxia-responsive nano-drug delivery system relying on ASP[13, 14].\u0026nbsp;However, the effect of ASP on ovarian cancer has not been reported.\u003c/p\u003e\n\u003cp\u003eIn this study, we investigated the in vitro effects of ASP on SKOV3 ovarian cancer cells, as well as cisplatin-resistant SKOV3/DDP cells. Our experiments demonstrated that ASP combined with DDP can effectively reverse the resistance of SKOV3/DDP cells to DDP. We also verified the efficacy of ASP combined with DDP against tumors in nude mice, as well as evaluated the safety of ASP in vivo. Furthermore, we utilized RNA-seq data to identify differentially expressed genes (DEGs) and analyzed them to identify key genes. The effects of these key genes on cell function were subsequently verified through experiments.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eSKOV3 and SKOV3/DDP cells were purchased from Shanghai Fuyu Biotechnology Co., LTD. McCoy '5a (Tianhang Bio, Hangzhou, China) with 10% Fetal Bovine Serum (FBS) (Service Bio, Wuhan, China)was cultured in an incubator with 37 ℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e. In addition, SKOV3/DDP cells need to be added 0.5ug/ml DDP (Meilun Bio Dalian, China) to maintain DDP resistance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell viability and combination index (CI)\u003c/h2\u003e \u003cp\u003eThe SKOV3 and SKOV3/DDP cells of the DDP group were subjected to varying concentrations of cisplatin (0 \u0026micro;g/mL, 1.5 \u0026micro;g/mL, 3 \u0026micro;g/mL, 6 \u0026micro;g/mL, 12 \u0026micro;g/mL, 24 \u0026micro;g/mL) and cultured in a 37℃, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. Meanwhile, for the ASP group, SKOV3 and SKOV3/DDP cells were treated with different concentrations of ASP (Batch Number: C23A6Y1, Yuanye Bio, Shanghai, China) (0 \u0026micro;g/mL, 50 \u0026micro;g/mL, 100 \u0026micro;g/mL, 150 \u0026micro;g/mL, 200 \u0026micro;g/mL, 250 \u0026micro;g/mL, 300 \u0026micro;g/mL) and also cultured in a 37℃, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 48 hours. In the ASP combined with DDP group, SKOV3/DDP cells were subjected to 10 \u0026micro;g/mL DDP concentration, followed by the addition of different concentrations (0 \u0026micro;g/mL, 50 \u0026micro;g/mL, 100 \u0026micro;g/mL, 150 \u0026micro;g/mL, 200 \u0026micro;g/mL, 250 \u0026micro;g/mL, 300 \u0026micro;g/mL) of ASP in each group. The cell viability assay was conducted using CCK8 (Wanlei Bio, Shenyang, China). Furthermore, we used CompuSyn software (CompuSyn Inc.) to determine the Combination index (CI). The study results indicated the following: CI\u0026thinsp;=\u0026thinsp;0.85 to 0.9 representing slight synergism; CI\u0026thinsp;=\u0026thinsp;0.7 to 0.85 representing moderate synergism; CI\u0026thinsp;=\u0026thinsp;0.3 to 0.7 representing synergism; CI\u0026thinsp;=\u0026thinsp;0.1 to 0.3 representing strong synergism; and CI\u0026thinsp;\u0026lt;\u0026thinsp;0.1 representing very strong synergism.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo xenograft assays\u003c/h2\u003e \u003cp\u003eThe study utilized female BALB/c nude mice obtained from Cavens Laboratory Animal Co., Ltd. The mice, which were 4 weeks old, were split into ten mice per group. To induce tumor formation, SKOV3/DDP cells were dissolved in 20 \u0026micro;L of phosphate-buffered saline (PBS) and then subcutaneously injected into the armpits of the mice. The growth of the tumors to 100 mm\u003csup\u003e3\u003c/sup\u003e was set as day 1. For the Vehicle (Veh) group, the nude mice were intraperitoneally injected with 50 mL/kg/d PBS. Meanwhile, the DDP group was intraperitoneally injected with 4 mg/kg DDP on day 1 and 8 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Following Hu Zhuang et al.'s research on restraining breast tumor growth through the intraperitoneal injection of 0.2 mg/kg/d ASP in nude mice [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], the ASP group mice were also intraperitoneally injected with 0.2 mg/kg/d ASP. The tumors of the mice in each group were monitored every three days to determine the tumor volume \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e.After 21 days, the mice were euthanized, and the tumors were collected for analysis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. The animal experiment was conducted following the Guidelines for the Care and Use of Laboratory Animals and approved by the Ethics Committee of Harbin Medical University (certificate number: KY2023-37)..\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eHistological analysis\u003c/h2\u003e \u003cp\u003eWe took tissue extraction and embedding sections from the liver, kidney, and heart of the killed nude mice. Hematoxylin (Solarbio, Shanghai, China) and eosin Y (Sangon, Shanghai, China) were used for staining. Histological sections were taken with a microscope (OLYMPUS, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell invasion assays\u003c/h2\u003e \u003cp\u003eThe Transwells (Labselect, Hefei, China) were mixed with Matrigel (Corning, USA) and placed into a 24-well plate, which was then placed in a 37℃ incubator for 2 hours to allow the Matrigel to solidify. Next, 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well were added to the upper chamber and 800 \u0026micro;l of culture medium, containing 10% FBS, was added to the lower chamber. After 24 hours, the transwells were removed, washed twice with PBS, fixed with 4% neutral paraformaldehyde (Aladdin, Shanghai, China) for 20 minutes, and stained with 0.1% crystal violet (Amresco, USA) for 5 minutes. Finally, cells on the outer surface of the membrane were observed with a microscope and the number of cells passing through the permeable membrane in the visual field was randomly counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq analysis\u003c/h2\u003e \u003cp\u003eSKOV3/DDP cells were treated with either DDP or ASP combined with DDP. RNA was then extracted from both groups of cells using TRIzol (TaKaRa Bio, Dalian, China). The experiment was repeated three times using independent biological samples. The high-throughput sequencing platform NovaSeq 6000 by Illumina was utilized to sequence the samples. Raw data were filtered and assessed for quality. The clean reads were mapped to the human genome (GENCODE download, GRCh38) for Inter-sample expression level and principal component analysis. The DESeq2 R package was employed to analyze the raw data, and the differential expression threshold for DEGs was set to P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and |log\u003csub\u003e2\u003c/sub\u003eFoldChange| \u0026gt; 1. DEGs were subjected to GO and KEGG analyses using David (v2022q2, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://david-d.ncifcrf.gov/\u003c/span\u003e\u003cspan address=\"https://david-d.ncifcrf.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eLentivirus infection\u003c/h2\u003e \u003cp\u003eThe sequence of GPX4 gene (NM_001039848.4, GENERAL Bio, Chuzhou, China) was linked to the pLVX-IRES-puro vector (Fenghui Bio, Changsha, China) and transfected into 293T cells. After 48 hours, the lentivirus supernatant was collected and used to transfect SKOV3/DDP cells for 12 hours. Subsequently, the cells were screened with puromycin for 7 days to select for GPX4 overexpression. The overexpression of GPX4 was confirmed using RT-qPCR and Western blotting.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eRT-qPCR\u003c/h2\u003e \u003cp\u003eRNA extracted from SKOV3/DDP cells in each group was obtained via TRIzol (TaKaRa Bio, Dalian, China). Then, mRNA was reverse-transcribed into cDNA with the BeyoRT\u0026trade; II cDNA First Strand Synthesis Kit (Beyotime, Shanghai, China). GAPDH was used as the internal parameter. The reaction system consisted of 1 \u0026micro;L cDNA and 10 \u0026micro;L SYBR Green Master Mix (Solarbio, Beijing, China), 0.5 \u0026micro;L of upstream and downstream primers each (GENERAL Bio, Chuzhou, China), and 8 \u0026micro;L ddH\u003csub\u003e2\u003c/sub\u003eO. The reaction conditions included an initial denaturation of 95\u0026deg;C for 5 min, denaturation of 95\u0026deg;C for 10 s, annealing of 60\u0026deg;C for 10 s, extension of 72\u0026deg;C for 1 min (40 cycles), and final extension of 1 min 30s at 72\u0026deg;C. The primer sequences used were GPX4 F, GAAGCAGGAGCCAGGGAGT and GPX4 R, CGCAGCCGTTCTTGTCG; β-actin F, GGCACCCAGCACAATGAA and β-actin R, TAGAAGCATTTGCGGTGG.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eEach group was lysed with cell lysis solution (Wanlei Bio, Shenyang, China). The BCA protein concentration assay kit (Wanleibio, Shenyang, China) was used to quantify the protein concentration of each group of cells. The proteins were isolated by 14% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Wanlei Bio, Shenyang, China) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). PVDF membrane was sealed with 5% skim milk for 1 hour and incubated overnight with primary anti-GPX4 (Abclonal, Wuhan, China) and anti-β-actin (Wanlei Bio, Shenyang, China) at 4℃. Sheep Anti-Rabbit IgG-HRP (Wanlei Bio, Shenyang, China) was added and incubated at 37℃ for 45min. Finally, ECL (Wanlei Bio, Shenyang, China) was added for exposure scanning, and Gel-Pro-Analyzer software was used to analyze the optical density of the strips.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFerroptosis determination\u003c/h2\u003e \u003cp\u003eFe\u003csup\u003e2+\u003c/sup\u003e, MDA, SOD, and GSH are indicators of ferroptosis. To measure the contents of these indicators, we used Ferrous Iron Colorimetric Assay Kit (Elabscience, Wuhan, China), MDA Colorimetric Assay Kit (Wanlei Bio, Shenyang, China), Total Superoxide Dismutase Activity Assay Kit (Wanlei Bio, Shenyang, China), and Reduced Glutathione Colorimetric Assay Kit (Wanlei Bio, Shenyang, China) following the manufacturer's instructions. We measured the absorbance and calculated the concentrations of Fe\u003csup\u003e2+\u003c/sup\u003e, MDA, SOD, and GSH in each cell group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eWe used GraphPad Prism 8.0 software to analyze the results of the experiments. The student t-test was used to compare the differences between two groups, while ANOVA was used to compare between three or more groups. Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003eASP and DDP can inhibit the proliferation of SKOV3 and SKOV3/DDP\u003c/h2\u003e\n \u003cp\u003eSKOV3 and SKOV3/DDP cells were treated with DDP (1.5µg/ml, 3 µg/ml, 6µg/ml, 12µg/ml, 24 µg/ml), and cell viability was measured 48 hours later. The results are shown below, SKOV3 groups: compared with the vehicle group, the viability of each group was 81.95%±5.37, 57.70%±7.66, 43.28%±7.09, 35.22%±4.07, 24.56%±3.03; SKOV3/DDP groups: Compared with the vehicle group, the viability of each group was 94.07%±4.36, 88.15%±10.89, 79.20%±9.10, 66.82%±7.92, 53.08%±7.58\u003cstrong\u003e(\u003c/strong\u003eFig. 1A\u003cstrong\u003e)\u003c/strong\u003e. The viability of each group decreased with increasing DDP concentration when compared to the vehicle group. Similarly, SKOV3/DDP groups showed a decrease in viability with increasing DDP concentration. Student t-test was used to compare the proliferative activity of DDP against SKOV3 and SKOV3/DDP cells, showing significant differences in sensitivity to DDP (P\u003c0.01). We also treated SKOV3 and SKOV3/DDP cells with ASP (50 µg/mL, 100 µg/mL, 150 µg/mL, 200 µg/mL, 250 µg/mL, 300µg/mL), and cell viability was measured 48 hours later. SKOV3 group: Compared with the vehicle group, the viability of each group was 98.95%±8.59, 97.03%±7.57, 94.96%±13.33, 90.37%±6.49, 82.54%±3.83, 76.33%±3.56. SKOV3/DDP group: Compared with the vehicle group, the viability of each group was 98.76%±8.13, 97.61%±6.73, 96.37%±6.56, 90.72%±5.05, 87.86%±0.64, 83.92%±1.41\u003cstrong\u003e(\u003c/strong\u003eFig. 1B\u003cstrong\u003e)\u003c/strong\u003e.Both SKOV3 and SKOV3/DDP groups showed a decrease in viability with increasing ASP concentration. Overall, ASP inhibited the proliferation of SKOV3 and SKOV3/DDP cells, with increasing inhibition at higher concentrations of ASP.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\"\u003e\n \u003ch2\u003eASP combined with DDP increases the sensitivity of SKOV3/DDP to DDP\u003c/h2\u003e\n \u003cp\u003eTo assess the efficacy of the combination of ASP and DDP on SKOV3/DDP cells, we calculated the combination index (CI) of ASP and DDP. Each group was supplemented with 10 µg/mL of DDP and varying concentrations of ASP. After 48 hours, cell viability was examined and compared to that of the control group. Cell viability decreased with increasing concentrations of ASP, with values of 75.74%±6.76, 70.39%±6.77, 59.96%±8.83, 56.90%±2.91, 51.95%±3.88, 47.47%±3.93, and 38.33%±4.87% \u003cstrong\u003e(\u003c/strong\u003eFig. 2A\u003cstrong\u003e)\u003c/strong\u003e. Using CompuSyn software, we calculated the CI for each group and obtained values of 1.06, 0.73, 0.71, 0.65, 0.60, and 0.47 \u003cstrong\u003e(\u003c/strong\u003eFig. 2B\u003cstrong\u003e)\u003c/strong\u003e. These results indicate that ASP and DDP exhibit a synergistic effect, except for ASP at a concentration of 50 µg/mL.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\"\u003e\n \u003ch2\u003eEffect of ASP combined with DDP on SKOV3/DDP in vivo, and the evaluation of safety\u003c/h2\u003e\n \u003cp\u003eThe tumor size and growth rate in nude mice were lower in the ASP combined with DDP group than in the other groups. ASP combined with DDP showed higher tumor inhibition rates (52.05%, 48.93%, and 73.99%) than ASP group, DDP group, and vehicle group \u003cstrong\u003e(\u003c/strong\u003eFig. 3C\u003cstrong\u003e)\u003c/strong\u003e. We collected liver, kidney, and heart tissue from the mice and used HE staining to assess the safety of ASP and DDP treatment in vivo. The results indicated that ASP combined with DDP had no significant effect on the morphology of the liver, kidney, or heart, suggesting that it is a safe treatment option \u003cstrong\u003e(\u003c/strong\u003eFig. 3D\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003eEffects of ASP combined with DDP on SKOV3/DDP cell invasion\u003c/h2\u003e\n \u003cp\u003eThe effects of the ASP group, the DDP group, and ASP combined with DDP group on SKOV3/DDP cell invasion were investigated using the Transwell method. The combined ASP and DDP group demonstrated lower average cell counts passing through the permeable membrane of the Transwell compared to the ASP and DDP groups. The ASP combined with DDP showed a stronger inhibition of SKOV3/DDP cell invasion \u003cstrong\u003e(\u003c/strong\u003eFig. 4A\u003cstrong\u003e\u0026amp;B)\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003eThe cell's transcriptome was sequenced to identify key genes\u003c/h2\u003e\n \u003cp\u003eTo investigate whether the combination of ASP and DDP can regulate key genes in SKOV3/DDP transcription process, we conducted transcriptome sequencing on two groups: ASP combined with DDP and DDP alone. First, the correlation analysis \u003cstrong\u003e(\u003c/strong\u003eFig. 5A\u003cstrong\u003e)\u003c/strong\u003e and principal component analysis (PCA) \u003cstrong\u003e(\u003c/strong\u003eFig. 5B\u003cstrong\u003e)\u003c/strong\u003e indicate that the obtained data is reliable and there are significant differences between the data of the DDP group and the ASP combined with DDP group. Next, using the defined threshold, we obtained 843 DEGs\u003cstrong\u003e(\u003c/strong\u003eFig. 5C\u003cstrong\u003e)\u003c/strong\u003e, among which 405 down-regulated genes and 438 up-regulated genes existed \u003cstrong\u003e(\u003c/strong\u003eFig. 5D\u003cstrong\u003e) (Table.1s)\u003c/strong\u003e.We then analyzed these DEGs by GO and KEGG. The GO enrichment analysis showed that Molecular Function (MF) was mainly enriched in structural constituent of chromatin, structural constituent of ribosome, and protein heterodimerization activity. Biological Processes (BP) were mainly concentrated in cytoplasmic translation, nucleosome assembly, and translation. Cellular Component (CC) was mainly concentrated in nucleosome and mitochondrial inner membrane \u003cstrong\u003e(\u003c/strong\u003eFig. 6A\u003cstrong\u003e\u0026amp;B) (Table.2s)\u003c/strong\u003e. KEGG enrichment analysis showed that DEGs were mainly concentrated in oxidative phosphorylation, tumor transcriptional regulation, and other pathways \u003cstrong\u003e(\u003c/strong\u003eFig. 6C\u003cstrong\u003e\u0026amp;D) (Table.3s)\u003c/strong\u003e. Previous studies by Xiaodong Wu et al. have found that the expression level of GPX4 in SKOV3/DDP cells is higher than in SKOV3 cells and that its increase is associated with the prognosis and drug resistance of patients[17]. After conducting a comprehensive analysis on both log\u003csub\u003e2\u003c/sub\u003e Fold Change ranking and P-value ranking, as well as reviewing relevant publications, it is postulated that GPX4 is a key gene involved in the regulation of SKOV3/DDP cells. Consequently, our subsequent experiment will be centered on the exploration of GPX4.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\"\u003e\n \u003ch2\u003eRTqPCR and western blot analysis of key genes GPX4\u003c/h2\u003e\n \u003cp\u003eWe treated SKOV3/DDP cells with ASP, DDP, and a combination of ASP and DDP. GPX4 expression was detected via both RT-qPCR \u003cstrong\u003e(\u003c/strong\u003eFig. 7A\u003cstrong\u003e)\u003c/strong\u003e and western blot \u003cstrong\u003e(\u003c/strong\u003eFig. 7B\u003cstrong\u003e\u0026amp;C)\u003c/strong\u003e. Our results showed that GPX4 was significantly down-regulated in the group treated with a combination of ASP and DDP. Furthermore, to investigate the role of GPX4 in SKVO3/DDP cells, we generated stable SKVO3/DDP cells that overexpress GPX4, referred to as SKOV3/DDP-GPX4 cells. We then compared the expression of GPX4 in SKOV3/DDP, SKOV3/DDP-NC (negative control group), and SKOV3/DDP-GPX4 cells using RT-qPCR \u003cstrong\u003e(\u003c/strong\u003eFig. 7D\u003cstrong\u003e)\u003c/strong\u003e and western blot \u003cstrong\u003e(\u003c/strong\u003eFig. 7E\u003cstrong\u003e\u0026amp;F)\u003c/strong\u003e. The data suggested that GPX4 was highly expressed in SKOV3/DDP-GPX4 cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\"\u003e\n \u003ch2\u003eASP combined with DDP inhibited SKOV3/DDP proliferation and invasion by inhibiting GPX4\u003c/h2\u003e\n \u003cp\u003eThe control group, SKOV3/DDP-NC group, and SKOV3/DDP-GPX4 group were treated with Veh, DDP, ASP combined with DDP, respectively, and the cell proliferation of each group was detected using CCK8. The results showed that the cell viability of the control group, SKOV3/DDP-NC group, and SKOV3/DDP-GPX4 group treated with ASP combined with DDP was significantly lower than that of the group treated with DDP alone \u003cstrong\u003e(Fig.\u0026nbsp;8A)\u003c/strong\u003e. Similarly, transwell assay was performed to assess the cell invasion ability, and it was observed that the cell invasion ability of the control group, SKOV3/DDP-NC group, and SKOV3/DDP-GPX4 group treated with ASP combined with DDP was significantly lower than that of the group treated with DDP alone \u003cstrong\u003e(Fig.\u0026nbsp;8B\u0026amp;C)\u003c/strong\u003e. These results indicate that ASP combined with DDP can effectively inhibit cell proliferation and invasion by suppressing GPX4.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\"\u003e\n \u003ch2\u003eASP combined with DDP can cause ferroptosis of SKOV3/DDP by inhibiting GPX4 expression of SKOV3/DDP\u003c/h2\u003e\n \u003cp\u003eTo investigate how ASP combined with DDP facilitates ferroptosis in SKOV3/DDP cells, we conducted experiments to determine the levels of Fe\u003csup\u003e2+\u003c/sup\u003e, MDA, SOD, and GSH. The control group, SKOV3/DDP-NC group, and SKOV3/DDP-GPX4 group were treated with Veh, DDP, and ASP combined with DDP. We observed a significant increase in Fe\u003csup\u003e2+\u003c/sup\u003e and MDA content in all groups following the treatment of ASP combined with DDP \u003cstrong\u003e(\u003c/strong\u003eFig. 9A\u003cstrong\u003e\u0026amp;B)\u003c/strong\u003e. Conversely, the levels of SOD and GSH were significantly reduced in all groups treated with ASP combined with DDP \u003cstrong\u003e(\u003c/strong\u003eFig. 9C\u003cstrong\u003e\u0026amp;D)\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAt present, drug resistance remains a significant cause of poor prognosis in individuals with cancer[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Research has indicated that drug resistance in tumors is connected to the tumor microenvironment, and traditional Chinese medicine may be able to reverse drug resistance by regulating the tumor microenvironment [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. ASP possesses multiple functions, such as anti-tumor capabilities, the regulation of cell microenvironment, and the treatment of gynecological diseases[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Thus, we hypothesize that ASP can reverse the drug resistance of ovarian cancer by regulating its microenvironment or substance metabolism. Initially, we conducted a verification experiment on SKOV3 and the sensitivity of SKOV3/DDP cells to DDP. The experiment demonstrated that the sensitivity of SKOV3/DDP cells to DDP was lower than that of SKOV3 cells. We also conducted experiments on whether ASP has an inhibitory effect on SKOV3 and SKOV3/DDP cells. The results indicated that ASP had an inhibitory effect on SKOV3 but had a poor inhibitory effect on SKOV3/DDP cells. To better understand whether the combination of two drugs has a synergistic effect, we calculated the combination index (CI) values of the two drugs[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The results showed that the two drugs had excellent synergistic effects. Additionally, we performed in vivo testing with the drugs. After subcutaneously forming tumors in nude mice, we intraperitoneally injected the drugs and periodically measured the condition of the tumors in the mice. The results showed that the combination of ASP and DDP had an excellent tumor inhibition effect. Furthermore, evaluating drug safety is crucial when considering drug clinical applications [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. It has been reported that ASP has antioxidant protection on cardiac cells [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and reduce liver fibrosis induced by ccl\u003csub\u003e4\u003c/sub\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].The liver, heart, and kidney conditions were evaluated in each group of nude mice, and no differences were found among them, demonstrating that ASP combined with DDP had good safety. Tumor invasion is related to drug resistance [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], so the effects of ASP, DDP, and ASP combined with DDP on SKOV3/DDP invasion were investigated. The results revealed that ASP combined with DDP could better inhibit SKOV3/DDP invasion. Next, we explored the specific mechanism by which ASP combined with DDP changes the resistance of SKOV3/DDP cells to DDP.\u003c/p\u003e \u003cp\u003eComparison of the RNA-seq data between the DDP group and the ASP combined with DDP group revealed the presence of numerous DEGs involved in transcription. GO [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and KEGG[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] analyses of the DEGs showed enrichment in metabolic processes and cellular transcriptional regulation. Specifically, cancer cells undergo different metabolic processes from normal cells[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], with increased glycolysis demonstrated by the Warburg effect [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and a dependence on rapid tricarboxylic acid (TCA) cycling to sustain proliferation [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The Warburg effect has also been linked to drug resistance [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Additionally, drug resistance in ovarian cancer has been associated with cellular transcriptional regulation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In conclusion, the analysis suggests a relationship between the combination of ASP with DDP and the regulation of drug resistance in SKOV3/DDP cells.\u003c/p\u003e \u003cp\u003eWe sequenced differentially expressed genes based on their log\u003csub\u003e2\u003c/sub\u003e fold change and p-value, and reviewed the relevant literature. According to previous studies, GPX4 expression is higher in SKOV3/DDP cells compared to SKOV3 cells, and inhibition of GPX4 can reduce drug resistance in resistant cells[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, we hypothesized that downregulation of GPX4, caused by the combination of ASP and DDP, is responsible for the decreased resistance of SKOV3/DDP cells.\u003c/p\u003e \u003cp\u003eIn subsequent experiments, SKOV3/DDP cells were stably transfected with lentivirus to overexpress GPX4, and relevant experiments were conducted. The results showed that overexpressing GPX4 in SKOV3/DDP cells increased their resistance to DDP, while combining ASP with DDP reversed DDP resistance.\u003c/p\u003e \u003cp\u003eStudies have shown that GPX4 is a crucial gene involved in regulating ferroptosis in cells [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Ferroptosis is a complex biological process characterized by a toxic accumulation of lipid peroxides on cell membranes[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. It has also been associated with tumor occurrence and development [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Specifically, Wang Y et al. demonstrated that the regulation of ferroptosis can affect the sensitivity of platinum-resistant ovarian cells to DDP[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The presence of Fe\u003csup\u003e2+\u003c/sup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]and MDA[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], which are products of membrane lipid peroxidation, plays a crucial role in detecting ferroptosis. Moreover, SOD [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and GSH [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] are also used as diagnostic criteria for ferroptosis in cells. Experimental results revealed that the combination of ASP and DDP treatment led to a significant increase in Fe\u003csup\u003e2+\u003c/sup\u003e and MDA levels, while SOD and GSH levels were significantly decreased. These changes indirectly indicate a reduction in GPX4 expression and an exacerbation of ferroptosis in SKOV3/DDP cells.\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBecause ASP is predominantly composed of glucose (Glc), galactose (Gal), arabinose (Ara), rhamnose (Rha), fucose (Fuc), xylose (Xyl), and galacturonic acid (GalUA)[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], we will conduct further investigations on the monosaccharide components in ASP in order to examine their impact on tumor. Additionally, our study reveals an interesting finding from the GO analysis, indicating an enrichment of the Cellular Component (CC) in the mitochondrial inner membrane. It is well-known that cellular ferroptosis induces alterations in mitochondria [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Mitochondria, being instrumental in cysteine-deprivation induced (CDI) ferroptosis, do not, however, play a significant role in inhibiting GPX4-induced ferroptosis[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. As a result, we question whether ASP combined with DDP possesses an alternative mechanism for regulating the ferroptosis of SKOV3/DDP cells, aside from its influence on GPX4. Moreover, it is worth noting that GPX4 expression is frequently controlled by Non-coding RNA [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. We hypothesize that the combination of ASP and DDP can regulate the GPX4 expression in SKOV3/DDP through non-coding RNA, thereby promoting an increase in SKOV/DDP ferroptosis. This aspect will also be one of our focal points of investigation.\u003c/p\u003e \u003cp\u003eDue to the intricate mechanism of ferroptosis in tumors, additional experiments will be conducted to determine the specific mechanism by which ASP combined with DDP inhibits GPX4 expression.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe discovered that ASP combined with DDP can regulate the transcription process of SKOV3/DDP, and it can inhibit the expression of the key gene GPX4 to promote the ferroptosis of SKOV3/DDP. This finding provides a theoretical basis for the clinical application of ASP.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eASP \u0026nbsp;Angelica Sinensis Polysaccharide\u003c/p\u003e\n\u003cp\u003eDDP \u0026nbsp;cisplatin\u003c/p\u003e\n\u003cp\u003eMDA \u0026nbsp;malondialdehyde\u003c/p\u003e\n\u003cp\u003eSOD \u0026nbsp; superoxide dismutase\u003c/p\u003e\n\u003cp\u003eGSH \u0026nbsp; Glutathione\u003c/p\u003e\n\u003cp\u003eHE \u0026nbsp;Hematoxylin-eosin\u003c/p\u003e\n\u003cp\u003eGPX4 \u0026nbsp;Recombinant Glutathione Peroxidase 4\u003c/p\u003e\n\u003cp\u003eRNA-seq \u0026nbsp;RNA-sequencing\u003c/p\u003e\n\u003cp\u003eDEGs \u0026nbsp;Differentially Expressed Genes\u003c/p\u003e\n\u003cp\u003eCCK8 \u0026nbsp;Cell Counting Kit-8\u003c/p\u003e\n\u003cp\u003ePBS \u0026nbsp;Phosphate Buffered Saline\u003c/p\u003e\n\u003cp\u003eFBS \u0026nbsp;Fetal Bovine Serum\u003c/p\u003e\n\u003cp\u003eCI \u0026nbsp;Combination Index\u003c/p\u003e\n\u003cp\u003ePVDF \u0026nbsp;polyvinylidene fluoride\u003c/p\u003e\n\u003cp\u003eVeh \u0026nbsp;Vehicle group\u003c/p\u003e\n\u003cp\u003eGO \u0026nbsp;Gene Ontology\u003c/p\u003e\n\u003cp\u003eKEGG \u0026nbsp;Kyoto Encyclopedia of Genes and Genomes\u003c/p\u003e\n\u003cp\u003eMF \u0026nbsp;Molecular Function\u003c/p\u003e\n\u003cp\u003eBP \u0026nbsp;Biological Processes\u003c/p\u003e\n\u003cp\u003eCC \u0026nbsp;Cellular Component \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMDA \u0026nbsp;Malondialdehyde\u003c/p\u003e\n\u003cp\u003eCDI \u0026nbsp;cysteine-deprivation induced\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperiment Design: Yaoxian Wang Weikang Guo. Experiment Operation:\u0026nbsp;Weikang Guo, Fei\u003c/p\u003e\n\u003cp\u003eLei,\u0026nbsp;Ruxin Zheng, Xinyao Zhao,Yunshun Tong.Data Statistics:\u0026nbsp;Yuze Gu,\u0026nbsp;Mengdi Yang,\u0026nbsp;Yunshun Tong. Article Written:\u0026nbsp;Weikang Guo,Wanyue Wang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWU JIEPING Medical Foundation Special Fund for Clinical Research (No.320.6750.2023-05-4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll datasets generated for this study are included in the article/ supplementary material, further inquiries can be directed to the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal study was reviewed and approved by the Ethics Committee of Harbin Medical University(certificate number: KY2023-37).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eManuscript is approved by all authors for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1Department of Gynecology, Harbin Medical University Cancer Hospital, Harbin,150081, China\u003c/p\u003e\n\u003cp\u003e2.\u0026nbsp;School of Basic Medical Sciences, Qiqihar Medical University, Qiqihar,161006,China\u003c/p\u003e\n\u003cp\u003e3. School of Science, Department of Biological Sciences, Xi\u0026rsquo;an Jiaotong-Liverpool University, Suzhou, 215123, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018, 68(6):394-424.\u003c/li\u003e\n\u003cli\u003eStewart C, Ralyea C, Lockwood S: Ovarian Cancer: An Integrated Review. Semin Oncol Nurs 2019, 35(2):151-156.\u003c/li\u003e\n\u003cli\u003eBuechel M, Herzog TJ, Westin SN, Coleman RL, Monk BJ, Moore KN: Treatment of patients with recurrent epithelial ovarian cancer for whom platinum is still an option. Ann Oncol 2019, 30(5):721-732.\u003c/li\u003e\n\u003cli\u003eMunoz-Galvan S, Carnero A: Targeting Cancer Stem Cells to Overcome Therapy Resistance in Ovarian Cancer. 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Eur J Cell Biol 2020, 99(1):151058.\u003c/li\u003e\n\u003cli\u003eFernandez-Acosta R, Hassannia B, Caroen J, Wiernicki B, Alvarez-Alminaque D, Verstraeten B, Van der Eycken J, Vandenabeele P, Vanden Berghe T, Pardo-Andreu GL: Molecular Mechanisms of Nemorosone-Induced Ferroptosis in Cancer Cells. Cells 2023, 12(5).\u003c/li\u003e\n\u003cli\u003eHe GN, Bao NR, Wang S, Xi M, Zhang TH, Chen FS: Ketamine Induces Ferroptosis of Liver Cancer Cells by Targeting lncRNA PVT1/miR-214-3p/GPX4. Drug Des Devel Ther 2021, 15:3965-3978.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ovarian cancer, traditional Chinese medicine, angelica sinensis polysaccharide (ASP), GPX4, ferroptosis","lastPublishedDoi":"10.21203/rs.3.rs-3312243/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3312243/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAngelica sinensis (Oliv.) Diels has been used for centuries in Chinese traditional medicine to treat gynecological ailments. Numerous studies indicate that Angelica sinensis polysaccharide (ASP), an extract from Angelica sinensis, can inhibit various forms of cancer. Nevertheless, the therapeutic potential of ASP for treating ovarian cancer remains largely unexplored.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study investigated cell proliferation and invasion, as well as the effects of a combination of ASP and DDP after tumor formation in nude mice. Tumor size was monitored, and HE staining was conducted on tissue samples. The identification of key gene GPX4 was performed via RNA-seq and bioinformatic analysis. GPX4 was overexpressed using lentivirus transfection, and its expression was evaluated via RT-qPCR and western blot. Additionally, the ferroptosis of cells was assessed through the measurement of Fe\u003csup\u003e2+\u003c/sup\u003e, malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results indicated a synergistic effect of ASP combined with DDP, leading to better inhibition of proliferation and invasion of SKOV3/DDP cells. Similarly, ASP combined with DDP demonstrated tumor proliferation inhibition in tumor-forming nude mice, while maintaining good safety. Bioinformatics analysis of 843 differentially expressed genes (DEGs) revealed that the key gene GPX4 played a significant role in the mechanism of action. Furthermore, the expression of GPX4 was inhibited by ASP combined with DDP, which resulted in SKOV3/DDP inhibition of proliferation and invasion. The study also demonstrated that ASP combined with DDP led to increased levels of Fe\u003csup\u003e2+\u003c/sup\u003e\u0026nbsp;and MDA, while decreasing levels of GSH and SOD, suggesting the promotion ferroptosis of SKOV3/DDP cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe combination of ASP and DDP has the ability to inhibit the proliferation and invasion of SKOV3/DDP cells. Additionally, inhibiting GPX4 expression in SKOV3/DDP cells promotes ferroptosis.\u003c/p\u003e","manuscriptTitle":"The combination of Angelica sinensis polysaccharide and cisplatin promotes ferroptosis in cisplatin-resistant ovarian cancer cells by regulating GPX4, thereby reversing their resistance to cisplatin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-09-06 01:16:49","doi":"10.21203/rs.3.rs-3312243/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a02804d1-f4bd-4b5b-9ddb-983bb019445b","owner":[],"postedDate":"September 6th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-10-04T00:29:17+00:00","versionOfRecord":[],"versionCreatedAt":"2023-09-06 01:16:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3312243","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3312243","identity":"rs-3312243","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}